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Molecular Phylogenetics and Evolution xxx (2015) xxx–xxx

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Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/ locate /ympev

Systematics and biogeography of the Hylarana frog (Anura: Ranidae)radiation across tropical Australasia, Southeast Asia, and Africa q

http://dx.doi.org/10.1016/j.ympev.2015.05.0011055-7903/� 2015 Elsevier Inc. All rights reserved.

q This paper has been recommended for acceptance by A. Larson.⇑ Corresponding author at: Division of Vertebrate Zoology, Department of

Herpetology, American Museum of Natural History, Central Park West at 79th St,New York, NY 10024, United States.

E-mail address: Laoliver927@gmail.com (L.A. Oliver).

Please cite this article in press as: Oliver, L.A., et al. Systematics and biogeography of the Hylarana frog (Anura: Ranidae) radiation across tropictralasia, Southeast Asia, and Africa. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.05.001

Lauren A. Oliver a,b,⇑, Elizabeth Prendini b,c, Fred Kraus d, Christopher J. Raxworthy b

a Richard Gilder Graduate School, American Museum of Natural History, Central Park West at 79th St, New York, NY 10024, United Statesb Division of Herpetology, American Museum of Natural History, Central Park West at 79th St, New York, NY 10024, United Statesc Department of Ecology, Evolution and Natural Resource Conservation, Rutgers University, New Brunswick, NJ 08901, United Statesd Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI 48109, United States

a r t i c l e i n f o a b s t r a c t

31323334353637383940414243

Article history:Received 29 January 2015Revised 29 April 2015Accepted 1 May 2015Available online xxxx

Keywords:PhylogeneticsTime calibrationOverwater dispersalTaxonomyLydekker’s LineWallace’s Line

4445

We present an inclusive molecular phylogeny for Hylarana across its global distribution, utilizing twomitochondrial and four nuclear gene regions for 69 of the 97 currently described species. We use phylo-genetic methods to test monophyly of Hylarana, determine relationships among ten putative subgenera,identify major clades, reconstruct biogeographic history, and estimate continental dispersal dates.Results support Hylarana as a monophyletic group originating approximately 26.9 MYA and comprisingeight clades that partly correspond to currently described subgenera plus two new groups. The Africanand Australasian species each form clades embedded within a paraphyletic Southeast Asian group. Weestimate that Africa and Australasia were colonized by Hylarana s.l. from SE Asia approximately 18.7and 10.8 MYA, respectively. Biogeographic reconstructions also support three separate colonizationevents in India from Southeast Asia. Examination of museum specimens identified morphologicalcharacters useful for delineating subgenera and species. We herein elevate all supported subgenera togenus rank and formally describe two new genera to produce a revised taxonomy congruent with ournew phylogenetic and biogeographic findings.

� 2015 Elsevier Inc. All rights reserved.

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1. Introduction

Hylarana is a diverse genus of ranid frogs of great systematicand biogeographic interest due to its broad distribution acrossAfrica, Southeast Asia, and Australasia. However, its phylogeny isstill poorly known, rendering global biogeographic studies prob-lematic. Crossing multiple biogeographic barriers, Hylarana is oneof a few amphibian genera that extends from Southeast Asia pastWallace’s Line to Sulawesi and the Philippines, and further, pastLydekker’s Line to Australasia (Fig. 1); and it is one of only twosoutheast Asian ranoid genera that has reached Africa, the otherbeing Chiromantis Peters, 1854 (Rhacophoridae). Hylarana is theonly amphibian genus to inhabit all three of these biogeographicregions, providing a unique opportunity to calculate timing ofcontinental colonization events. Other terrestrial vertebrate

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families have a similar distribution across Australasia, Asia, andAfrica, such as Varanidae (monitor lizards), Pteropodidae (mega-bats), and Pythonidae (pythons).

There are currently 97 described species of Hylarana s.l., with 11in Africa, 73 in Southeast Asia, and 13 in Australasia (primarilyNew Guinea, with only H. daemeli reaching the northern portionof Australia) (Frost, 2014). The species diversity in Hylarana isundoubtedly underestimated; multiple molecular studies revealthat when widespread species are examined at the populationlevel, these ‘‘species’’ actually contain multiple deeply divergentand independently evolving lineages, some of which are not mono-phyletic, e.g., H. arfaki, H. aurantiaca, H. chalconota, H. flavescens, H.signata, and H. temporalis (Biju et al., 2014; Brown and Guttman,2002; Brown and Siler, 2014; Donnellan et al., 2010; Inger et al.,2009; Stuart et al., 2006; Zainudin and Sazali, 2012). An increasingnumber of described species in Hylarana, combined with its largegeographic range, has led to regional studies of limited and incon-sistent sampling for both molecular and morphological data. Thecontent of the genus has also been revised, based on comparativestudies that include other closely related ranid genera such asAmolops, Babina, Glandirana, Huia, Meristogenys, Odorrana, and

al Aus-

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Fig. 1. Distribution of Hylarana s.l. in Africa, Asia, and Australasia. Range based on occurrence records by islands and/or countries in Frost (2014). The dotted red line betweenSoutheast Asia and Australasia denotes Lydekker’s Line. The green line denotes Wallace’s Line, following Mayr (solid) and Huxley (stippled). Photo of Papurana papua by F.Kraus (BPBM 16325).

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Sanguirana; Glandirana and/or Sanguinrana form the sister taxon toHylarana s.l. (Bossuyt et al., 2006; Che et al., 2007; Dubois, 1992;Fei et al., 1990, 2010; Frost et al., 2006; Fuiten et al., 2011;Kurabayashi et al., 2010; Pyron and Wiens, 2011; Stuart, 2008;Wiens et al., 2009).

On the basis of phenetic similarity, Dubois (1992) erected eightsubgenera within Hylarana (detailed in Table 1), which he explic-itly stated were preliminary hypotheses. These are Amnirana(Africa); Chalcorana, Humerana, Pulchrana, Sanguirana, andSylvirana (Southeast Asia); and Papurana and Tylerana(Australasia). Based on the species originally assigned to these sub-genera, some (e.g., Sylvirana) have been found to be paraphyletic

Table 1Summary of current taxonomy for each subgenus with original author, type species, whethelevated to generic status (G) or synonymized (S).

Subgenus Description Type species

Abavorana This study Limnodytes luctuosus Peters, 1871Amnirana Dubois (1992) Hylarana amnicola Perret, 1977Boulengerana Fei et al. (2010) Rana guentheri Boulenger, 1882Chalcorana Dubois (1992) Hyla chalconotus Schlegel, 1837Humerana Dubois (1992) Rana humeralis Boulenger, 1887Hydrophylax Fitzinger (1843) Rana malabarica Tschudi, 1838Hylarana Tschudi (1838) Hyla erythraea Schlegel, 1837Indosylvirana This study Rana flavescens Jerdon, 1853Papurana Dubois (1992) Rana papua Lesson, 1826Pulchrana Dubois (1992) Polypedates signatus Günther, 1872Sylvirana Dubois (1992) Lymnodytes nigrovittatus Blyth, 1856Tenuirana Fei et al. (1990) Rana taipehensis Van Denburgh, 1909Tylerana Dubois (1992) Rana jimiensis Tyler, 1963

Please cite this article in press as: Oliver, L.A., et al. Systematics and biogeogrtralasia, Southeast Asia, and Africa. Mol. Phylogenet. Evol. (2015), http://dx.do

by molecular analyses having moderately complete taxonomicsampling (Che et al., 2007; Frost et al., 2006). A further three gen-eric names are currently synonymized within Hylarana:Hydrophylax (Fitzinger, 1843), Tenuirana (Fei et al., 1990), andBoulengerana (Fei et al., 2010). The need to partition Hylarana intomultiple genera has been previously suggested based on phy-logeny, morphology and geography (Biju et al., 2014; Brown andGuttman, 2002; Donnellan et al., 2010; Dubois, 1992). However,such taxonomic revision has not yet been undertaken, most likelybecause previous studies were geographically restricted, employeduneven taxonomic sampling, or contained non-overlapping sets oftaxa. There are also numerous problems with incorrect species

er the type species was included in this analysis, and whether the subgenus is being

Included in analysis Clade in phylogeny Taxonomic status

Yes A GYes D GYes H2 SYes C GYes E GGenBank I GYes F GGenBank G GYes J GYes B GYes H GYes F2 SYes J2 S

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determinations for tissues in museum collections, and ‘‘wide-spread’’ taxa that are probably not conspecific across their entirerange. The lack of a complete and robust identification key to thegenus, combined with a large number of species and taxonomicuncertainty, makes a large-scale genetic approach with dense tax-onomic sampling appropriate to identify clades within Hylarana.

Here, we present the most comprehensive molecular phylogenyto date for Hylarana so as to test the monophyly of the genus,investigate validity and relationships of subgenera, and assign pre-viously unstudied species to clades. We also present biogeographicreconstructions to examine the history of dispersal of Hylaranaacross major biogeographic regions that other amphibians havefailed to cross. Lastly, we present a time-calibrated phylogeny totest the timing of major dispersal events across continents.

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2. Materials and methods

2.1. Tissue and voucher sampling

We obtained tissue samples from existing museum collectionsof the American Museum of Natural History, New York (AMNH);Bernice Pauahi Bishop Museum, Honolulu (BPBM); CaliforniaAcademy of Sciences, San Francisco (CAS); Field Museum ofNatural History, Chicago (FMNH); Louisiana State UniversityMuseum of Natural Science, Baton Rouge (LSUMZ); Museum ofVertebrate Zoology, Berkeley (MVZ); National Museum of NaturalHistory (USNM), Washington, D.C.; Port Elizabeth Museum, PortElizabeth (PEM); Royal Ontario Museum, Toronto (ROM);Museum für Naturkunde, Berlin (RG/ZMB); as well as some tissuesfrom private collections (Appendix A). The dataset also includesGenBank sequences from studies providing initial species descrip-tions or that experts identified on Hylarana from that geographicregion. Final analyses included a total of 160 individuals(Appendix A): we sequenced 97 during this study and obtained63 from GenBank (Biju et al., 2014; Bossuyt et al., 2006; Brownand Siler, 2014; Che et al., 2007; Hasan et al., 2012; Jeong et al.,2013; Matsui and Hamidy, 2012; Stuart, 2008; Stuart et al.,2006; van der Meijden et al., 2005). A total of 69 of 97 describedspecies of Hylarana s.l. are included, with 53 of 73 species fromSoutheast Asia, 5 of 11 from Africa, and 11 of 13 fromAustralasia. Outgroup taxa include species from the closely relatedgenera Babina, Glandirana, Odorrana, and Sanguirana. We used Ranajaponica as the outgroup for all phylogenetic analyses.

The type species for each subgenus (Table 1 and Appendix A) isincluded in our dataset, as described by Fitzinger (1843), Fei et al.(1990), Dubois (1992), and Fei et al. (2010). For Humerana(H. humeralis) we used a sample from Biju et al. (2014), which theylabeled as ‘‘Hylarana cf. humeralis.’’ Frost et al. (2006) did notinclude any species allocated to Humerana in their phylogeneticanalysis, and they kept this subgenus separate from Hylarana s.l.because they could not assess its status. Multiple studies sincethen have suggested that Humerana should be subsumed underHylarana (Bortamuli et al., 2010; Hasan et al., 2012; Matsui et al.,2005; Pyron and Wiens, 2011), and we here treat all thesesubgeneric names as hypotheses to be tested.

We excluded all museum tissue samples of ‘‘Hylarana’’ whenpreliminary analyses did not group the sample within Hylaranaor any of the above-mentioned closely related outgroup generaand when voucher examination confirmed the misidentification.We resolved identity of vouchers of non-monophyletic terminalsbearing the same taxon designation from various institutions by:(a) checking available voucher specimens against taxon descrip-tions, and renaming as ‘‘sp.’’ any that did not match that descrip-tion nor could be identified to another species; (b) examininglocality data to determine whether the individual was within the

Please cite this article in press as: Oliver, L.A., et al. Systematics and biogeogrtralasia, Southeast Asia, and Africa. Mol. Phylogenet. Evol. (2015), http://dx.do

species’ known range, and using proximity to the type locality asa factor; (c) expertise of the person determining the sample atthe tissue’s depository. In many cases, an expert who had recentlypublished on a particular species group had identified some of thesamples used here, and we favored that identification over anony-mous ones. In most cases, misidentified material had beenassigned to widespread taxa with older names, e.g., H. albolabris,H. maosonensis, H. nigrovittata, H. signata, H. taipehensis. In the caseof H. nigrovittata in particular, previous studies have noted extremegeographic variation, suggesting cryptic diversity and the possibil-ity that there may be at least five species in the ‘‘nigrovittata’’ com-plex (Gawor et al., 2009; Matsui et al., 2001; Ohler et al., 2002). Wethus consider the stated identity of our ‘‘H. nigrovittata’’ samples tobe preliminary. We retained all sequence data for these unidenti-fied but vouchered samples and included them in the analysis,listed as ‘‘Hylarana (subgenus) sp.’’ (in Appendix A), for future tax-onomic study.

We used voucher specimens with molecular sequence data,supplemented with additional vouchers from the AMNH collection,to identify shared morphological characteristics of clades identifiedin the molecular phylogeny.

2.2. DNA isolation, amplification, and sequencing

We extracted whole genomic DNA from liver or muscle tissueusing the Qiagen DNeasy Blood and Tissue Kit (Valencia,California, USA). We sequenced two partial mitochondrial generegions (16S rRNA, Cytochrome b) and four partial nuclear generegions – C-X-C chemokine receptor type 4 (CXCR4),recombination-activating gene 1 (RAG1), nuclear proto-oncogenecellular myelocytomatosis exon 2 (C-myc 2), and Tyrosinase exon1(Tyr) – for all samples. We used published primers for Hylaranaor closely related genera (Bossuyt and Milinkovitch, 2000; Paulyet al., 2004; Shimada et al., 2011; Wiens et al., 2005). Gene regions,primer pairs, annealing temperatures, and basepair lengths arelisted in Table 2. We amplified target gene regions using standardPCR protocols and cleaned amplicons using Agencourt AMPure XPon a Beckman Coulter Biomek FX robot. We cycle sequenced puri-fied amplicons in both directions with a BigDye v. 3.1 TerminatorSequencing Kit (Applied Biosystems, Foster City, CA, USA) andsequenced on an Applied Biosystems Inc. Prism™ 3730xl auto-mated sequencer. We assembled and edited complementarystrands using Geneious v. 6.1 (Biomatters Inc.; www.geneious.-com) and aligned in MAFFT v. 7.0 (Katoh and Standley, 2013).We deposited all sequences in GenBank and accession numbersare: KR264033–KR264511.

2.3. Phylogenetic analysis

We used maximum likelihood (ML) and Bayesian inference onthe concatenated dataset for all six loci as well as for individualgene trees. For both analyses, we tested a variety of models andpartitioning strategies in jModelTest v. 2 (Posada, 2008) andPartitionFinder v. 1.1 (Lanfear et al., 2012), respectively, using theBayesian Information Criterion (BIC; Table 2) to determine the bestmodel. Partitioning strategies included by locus and by codon posi-tion for the five protein coding genes (Cyt b, C-myc 2, CXCR4, RAG1,and Tyr). We implemented maximum likelihood analysis in RAxMLv. 7.0 (Stamatakis, 2006) under default parameters and assessedbranch support with 1000 thorough bootstrap replicates. We alsogenerated individual gene trees for each locus in RAxML usingthe same parameters. We implemented Bayesian analyses inMrBayes v. 3.2 (Huelsenbeck and Ronquist, 2001; Ronquist andHuelsenbeck, 2003) on CIPRES Science Gateway (www.phylo.org/sub_sections/portal/) for two independent runs, each with fourchains, and run for 25 million generations with sampling every

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Table 2List of loci sequenced, PCR primers, length, and best-fit model. Partitioning scheme was recovered as divided by gene region.

Gene region Primer Sequence (50–30) Length (bp) Annealing temp Citation Best fit model

16S 12sm GGCAAGTCGTAACATGGTAAG 1512 51 Pauley et al. (2004) GTR + I + G16sd CTCCGGTCTGAACTCAGATCACGTAG

Cyt b cytb-c CTACTGGTTGTCCTCCGATTCATGT 567 51 Bossuyt and Milinkovich (2000) GTR + I + GCB-J-10933 TATGTTCTACCATGAGGACAAATATC

C-myc exon 2 cmyc1U GAGGACATCTGGAARAARTT 422 49 Wiens (2005) HKY + I + Gcmyc-ex2dR TCATTCAATGGGTAAGGGAAGACC

CXCR exon 4 CXCR4-B ATCATTGGCAATGGAYTRGT 650 50 Biju and Bossuyt (2003) HKY + I + GCXCR4-G AGGCAACAGTGGAARAANGC

Rag exon 1 Rag1_1F GCMTTGCTSCCRGGGTATCA 750 50 Shimada et al. (2011) HKY + I + GRag1_2R TCAATGGACGGAAGGGTTTCAATAA

Tyrosinase exon 1 Tyr1A AGGTCCTCTTRAGCAAGGAATG 597 57 Bossuyt and Milinkovich (2000) GTR + I + GTyr 1G TGCTGGGCRTCTCTC-CARTCCCA

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1000 generations. We unlinked substitution models, relative ratesof substitution, transition/transversion ratio, and stationarynucleotide frequency parameters for all partitions, and set rate pri-ors to variable to allow differing substitution rates among parti-tions. We left all other parameters at default settings. Weassessed convergence by examining (1) the potentialscale-reduction factors in MrBayes and (2) the traces of all param-eters and the effective sample sizes in Tracer v. 1.6 (Rambaut et al.,2014). We discarded the first 25% of samples as burn-in.

We also created a reduced matrix of 95 terminals for both MLand Bayesian analyses with one individual per species (based onthe sample with the most loci successfully sequenced), including‘‘species’’ left unidentified (‘‘sp.’’) after we examined voucher spec-imens (Appendix A). We used this reduced matrix in both the bio-geographic and time-calibrated analyses. This approach limited theamount of missing data in the phylogenetic analyses.

We also attempted to account for the differences between genetrees and species trees using coalescent analysis (Degnan andRosenberg, 2009, 2006; Edwards, 2009; Edwards et al., 2007;Maddison, 1997). However, due to the large number of terminals,combined with the amount of missing data for some taxa (e.g.,the GenBank samples), species tree analyses would not convergeor produce bifurcating trees (in the case of maximum likelihoodspecies tree analyses).

2.4. Biogeographic reconstructions

To examine the biogeographic history of Hylarana s.l., we imple-mented (1) maximum likelihood ancestral reconstruction (MLSAR)in Mesquite v. 2.74 (Maddison and Maddison, 2010), (2) StatisticalDispersal-Vicariance Analysis (S-DIVA) in RASP v. 3.0 (Yu et al.,2010, 2014), and (3) Dispersal-Extinction Cladogenesis (DEC)(Ree et al., 2005), also in RASP, to determine ancestral ranges foreach node. We defined geographic areas of occurrence as:A = sub-Saharan and central Africa; B = India, Nepal, and SriLanka; C = Southeast Asia, (Myanmar, Thailand, Cambodia,Vietnam, Laos, China, Malaysia, Java, Sumatra); D = Sulawesi;E = Phillipines; F = Australasia. We defined regions based onwell-established biogeographic and continental boundaries (e.g.,Lydekker’s Line, Wallace’s Line, Africa, Sunda Shelf, etc.). For allbiogeographic analyses, we used the MrBayes reduced concate-nated tree.

2.5. Time-calibration

Fossil taxa that can be unambiguously assigned to Ranidae aresparse, and there are currently no known fossils of Hylarana s.l.However, we calibrated the tree with two calibration points: (1)one fossil calibration based on the earliest fossil remains ofEuropean water frogs, Pelophylax (Rage and Rocek, 2003), and (2)

Please cite this article in press as: Oliver, L.A., et al. Systematics and biogeogrtralasia, Southeast Asia, and Africa. Mol. Phylogenet. Evol. (2015), http://dx.do

one published divergence date between Limnonectes kochangaeand the remaining ranids (Roelants et al., 2007). Therefore, for thisanalysis, we included GenBank sequence data for Meristogenys kin-abaluensis and Rana temporaria to calibrate the phylogeny with thewater frog fossil, as in Roelants et al. (2007). Both mitochondrialgene regions and three of the four nuclear loci (with the exceptionof C-myc 2) used in this study were available for M. kinabaluensisand R. temporaria. Additionally, we sequenced Limnonectes kochan-gae for the root and used the published divergence date fromRoelants et al. (2007) for calibration. We used BEAST v. 1.8(Drummond et al., 2012; Heled and Drummond, 2010) to calculatedivergence dates. We constrained monophyly for (1) Hylarana, and(2) all taxa but Limnonectes. Otherwise, we used BEAST to estimatethe topology independently so as not to interfere with datingestimates.

We estimated divergence times using an uncorrelated lognor-mal clock with a CTMC Rate Reference prior separately for eachgene partition and a birth–death incomplete-sampling tree prior(Gernhard, 2008) for 100 million generations, with the first 30%of samples being discarded as burn-in. We evaluated root datesusing a lognormal prior from 60 to 81 MY before present to cali-brate the phylogeny based on the same previously published data-set (Roelants et al., 2007). We set the tmrca (time of most recentcommon ancestor) for M. kinabaluensis and R. temporaria using alognormal prior between 28.6 and 40.5 MY before present (Rageand Rocek, 2003; Roelants et al., 2007). For more recent coloniza-tion events (e.g. before 15 MYA), we calculated genetic distancevalues for cytochrome b within and between clades in Mega 5.0(Tamura et al., 2011) to corroborate divergence time estimatesassuming a rate of �1% divergence per lineage per million years.We divided a sequence distance value by the inferred age of thecorresponding node; therefore, we used a value of approximately2 as an indicator of a more robust time estimate.

3. Results

3.1. Phylogenetic analyses and taxonomy

The final aligned length of the combined mitochondrial andnuclear dataset was 4498 bp. The partitioning scheme andbest-fit models for nucleotide substitution are provided inTable 2. Once we examined voucher specimens and re-identifiedor removed misidentified individuals from the dataset, the con-catenated analyses (Fig. 2) recovered a monophyletic Hylarana s.l.(64 ML bootstrap, 0.93 BPP). We found strong support (100 MLbootstrap, 1.00 BPP) for the monophyly of the Australasian groupof species and the African group (94 ML bootstrap, 1.00 BPP).However, both are embedded within a paraphyletic Asian group.Relationships among Amnirana, Chalcorana, and Pulchrana areincongruent between the maximum likelihood and Bayesian

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Fig. 2. Concatenated tree of Hylarana s.l. based on maximum likelihood and Bayesian inference of two mitochondrial and four nuclear gene regions. The tree is the consensustopology from the reduced matrix Bayesian analysis and is rooted with Rana japonica. Outgroup taxa are in gray and ingroup taxa (Hylarana s.l.) in black. Names on terminalscorrespond to the generic level taxonomy proposed in this paper. Node-support values are maximum likelihood bootstraps (above) and Bayesian posterior probabilities(below), respectively. Black circles on nodes indicate ML bootstrap P75 and BPP P0.95. White circles indicate BPP P0.95. Black squares indicate ML bootstrap P50 and BPPP0.75. White squares indicate ML bootstrap P75. Nodes that have a BPP <0.75 and ML bootstrap <50 are not listed. Bold terminals with an asterisk (⁄) are the type species forgenera proposed in this paper. Letters A–J represent clades corresponding to the putative (and newly recognized) genera recovered in both maximum likelihood and Bayesiananalyses. (A) Abavorana, gen. nov. (B) Pulchrana, (C) Chalcorana, (D) Amnirana, (E) Humerana (F) Hylarana s.s., (F2), Tenuirana, (G) Indosylvirana, gen. nov. (H) Sylvirana, (H2)Boulengerana, (I) Hydrophylax, (J) Papurana, (J2) Tylerana. Clade B is split into two clades (BA and BB) based on morphological differences.

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analyses, with the ML analysis having very low support (i.e., 19 MLbootstrap) and the Bayesian analysis good or strong support.Additionally, although some species-level relationships are

Please cite this article in press as: Oliver, L.A., et al. Systematics and biogeogrtralasia, Southeast Asia, and Africa. Mol. Phylogenet. Evol. (2015), http://dx.do

incongruent, the species composition of major clades areconsistent across analyses. Hylarana luctuosa is recovered as thesister taxon to all other Hylarana s.l. In both analyses,

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Glandirana + Sanguirana are recovered as the sister group toHylarana s.l., although our analysis did not support their reciprocalmonophyly. However, the support for this topological arrangementis low and based on limited GenBank sample data.

Our analyses retrieved each of Chalcorana and Humerana (asdescribed by Dubois, 1992) as monophyletic (Appendix A). Theother phenetically based subgenera described by Fei et al. (1990),Dubois (1992), and Fei et al. (2010) are all paraphyletic, as origi-nally suspected by these authors. However, it is possible to rede-fine most of these taxa to be monophyletic around theirrespective type species (Appendix A). Pulchrana contained thesame species as Dubois (1992) originally assigned to it, with theexception of H. luctuosa, which was consistent with Pyron andWiens (2011). Furthermore, we found Pulchrana to consist of twomorphologically diagnosable subclades (BA and BB). African spe-cies grouped into a single clade around H. amnicola, the type spe-cies of Amnirana. We recovered Hylarana nicobariensis, aSoutheast Asian species previously assigned to Sylvirana byDubois (1992), as the sister taxon to all African species, and wetransfer it to Amnirana. Hylarana malabarica, the type species ofHydrophylax (Fitzinger, 1843), groups with H. leptoglossa and H.gracilis (both assigned to Sylvirana by Dubois (1992)) in both anal-yses (98 ML bootstrap, 1.00 BPP). The analyses also recoveredmonophyly of Tylerana, but this clade was embedded withinPapurana. Dubois (1992) described both Tylerana and Papurana,and subsequently gave priority to Papurana (Dubois, 1999:Table 2). We retrieved Tenuirana (H. macrodactyla and H. taipehen-sis) as paraphyletic and embedded within the subgenus Hylarana,which has precedence. Phylogenetic analyses did not supportSylvirana as described by Dubois (1992). Rather, the species thatDubois (1992) placed in Sylvirana grouped primarily into twoclades that are not sister taxa. The nominate clade (formed aroundthe type species, H. nigrovittata) is the sister taxon to(Hydrophylax + Papurana). The monotypic Boulengerana (H. guen-theri) was embedded within the nominate clade of Sylvirana, andSylvirana takes precedence over that name. Remaining speciesoriginally assigned to Sylvirana by Dubois (1992) grouped into anunnamed clade comprising the H. aurantiaca group, H. flavescensgroup, H. intermedia, H. milleti, H. montana, and H. temporalis group,which is sister to (Sylvirana (Hydrophylax + Papurana)). We there-fore synonymize the names Boulengerana, Tylerana and Tenuiranabased on our phylogenetic results, raise remaining redefined sub-genera to generic rank (see Section 4.3), and describe two newclades to accommodate the unnamed taxa.

3.2. Morphology of voucher specimens

We provide a summary of morphological character states thatserve to distinguish among clades (Table 3). Clade names corre-spond to those depicted in Fig. 2. We also include additional diag-nostic characters of described subgenera as provided by Dubois(1992).

3.3. Biogeographic reconstructions and time calibration

MLSAR, S-DIVA, and DEC all support a Southeast Asian origin forHylarana s.l. (Fig. 3, Table 4). The analyses also support single col-onization events into Africa and into Australasia from SoutheastAsia. There is strong support for three separate dispersal eventsfrom Southeast Asia to India/Nepal/Sri Lanka by the Hydrophylax,Hylarana s.s., and Indosylvirana gen. nov. clades. Colonization ofSulawesi and Philippines is more ambiguous, with historic rangeestimations unable to distinguish between Southeast Asia andSulawesi, or between Southeast Asia and the Philippines.

The topology from the BEAST time-calibrated tree (Fig. 4)mostly agrees with the MrBayes and RAxML phylogenies except

Please cite this article in press as: Oliver, L.A., et al. Systematics and biogeogrtralasia, Southeast Asia, and Africa. Mol. Phylogenet. Evol. (2015), http://dx.do

for some species relationships within genera (e.g., withinChalcorana, Papurana and Pulchrana). Babina is recovered as withinthe sister taxon to the ingroup in the BEAST analysis, but the sup-port is very low. The relationship between clades of Hylarana s.l. iscongruent with the MrBayes concatenated phylogeny and iswell-supported. Papurana colonized Australasia approximately10.8 MYA and Amnirana colonized Africa approximately 18.7MYA. The mean group genetic distance for cytochrome b withinPapurana is 20.5%; when divided by the age of the node (in millionsof years) this gives a value of 1.9. India was colonized at least threetimes, approximately 16 MYA by Indosylvirana gen. nov. and 8.8MYA by Hydrophylax. The mean group genetic distance for cyto-chrome b within Indian Hydrophylax was 18.9%; when divided bythe age of the node this gives a value 2.1. Hylarana tytleri alsoreaches India, but we did not have accurately identified speciesfrom India to test colonization time. We found the root age ofHylarana s.l. to be 26.9 MYA.

4. Discussion

4.1. Continental colonization and diversification

The relationships among deep nodes of the tree for Hylarana s.l.were poorly supported in the ML analyses, but the Bayesian resultsprovided much stronger support. Previous studies with reasonabletaxonomic sampling have also had trouble resolving relationshipsamong deeply divergent clades in Hylarana s.l., retrieving low sup-port or no support at all (Pyron and Wiens, 2011; Stuart, 2008;Wiens et al., 2009). There could be multiple reasons why thisincongruence is replicated across multiple studies. A combinationof the age of the nodes and an early, rapid diversification asHylarana s.l. dispersed and colonized multiple continents wouldmake it difficult to resolve relationships (Fishbein et al., 2001;Fishbein and Soltis, 2004; Rokas et al., 2005). Regardless, our anal-yses strongly support monophyletic African (Amnirana) andAustralasian (Papurana) groups, indicating single colonizations ofboth biogeographic regions.

Australasian species had been hypothesized by Dubois (1992)to comprise two separate groups (Papurana and Tylerana), possiblydue to the morphological disparity of H. jimiensis from most otherspecies. New Guinean species can be extremely large for Hylaranas.l. (e.g., H. supragrisea and H. arfaki) and stand in some contrast toother species in the clade. This is possibly due to the availability ofunoccupied niches (large, semi-aquatic, terrestrial frogs were pre-viously absent from New Guinea) in the newly colonized region.Previous molecular studies did not sample sufficient species diver-sity to test the monophyly of Papurana or Tylerana. The coloniza-tion age estimate of New Guinea (Australasia) around 10–11 MYA corresponds with the docking of the Vogelkop Peninsulaonto New Guinea from the west, which occurred approximately10 MYA (Baldwin et al., 2012; Davies et al., 1997; Polhemus,2007). There has also been subsequent overwater dispersal of asingle species to the Solomon and Bismarck Islands (Fig. 3, Table 4).

Amnirana colonized Africa approximately 18–19 MYA. The sis-ter taxon of the African species was found to be H. nicobariensisfrom Southeast Asia (Nicobar Islands, Peninsular Thailand, Java,Sumatra, Borneo, and Philippines). This disjunct relationship sug-gests the possibility of a direct Indian Ocean dispersal event tosub-Saharan Africa. It is also possible that the current distributionis relictual due to extinction from the dry intervening regions.

We have estimated times of dispersal for two migrations intoIndia (e.g., Indosylvirana gen. nov. at 16 MYA and Hydrophylax at6–9 MYA). However, Humerana (e.g., H. humeralis), Hylarana s.s.(e.g., H. tytleri), and Sylvirana (H. nigrovittata) also occur in India,suggesting at least three more instances of colonization in India.

aphy of the Hylarana frog (Anura: Ranidae) radiation across tropical Aus-i.org/10.1016/j.ympev.2015.05.001

Table 3Summary of morphological diagnoses for each genus. Clade names correspond to Figs. 2–4 and descriptions in Section 4.3.

Clade A Abavorana Clade B Pulchrana Clade CChalcorana

Clade D Amnirana Clade EHumerana

Clade FHylarana

Clade GIndosylvirana

Clade H Sylvirana Clade IHydrophylax

Clade J Papurana

Posterior part ofabdominal skin

Granular BA is smooth andBB is granular

Granular Smooth or granular Smooth orslightlywrinkled

Smooth orslightlywrinkled

Granular orwrinkled

Smooth orgranular

Smooth orgranular

Smooth

Length of 1st versus2nd finger

1st > 2nd 1st P 2nd 1st 6 2nd 1st P 2nd 1st > 2nd 1 = 2 1 > 2 1 P 2 1 > 2 1 > 2

(Width of disc onFinger 3)/(Width ofFinger 3)

1–1.5 1.2–1.7 2–3.5 1–1.8 1–1.2 1.2–1.7 1.4–2 1.2–1.9 1–1.5 1.5–2

(Width of disc on Toe4)/(Width of Toe 4)

1–1.5 1–1.7 1.5–2 1–1.8 1–1.2 1–1.7 1.5–2 1–1.9 1–1.5 1.3–2

Dorsolateral folds:texture

Indistinct Fine or warty andpoorly developed

Thin or made upof a line of warts

Absent to extremelywell-developed (A.galamensis)

Complete andthin to well-developed

Well-developed

Thin andwell-defined

Medium andwell-developed

Thick and well-developed

Fine andgranular withasperities toabsent

Dorsolateral folds:color

May be white oryellow

Pale or brightcoloration, or asdorsum

Generallycolored asdorsum

Variable Palecoloration

Pale Differentialcoloration todorsum

Pale or samecoloration asdorsum

Differentialcoloration todorsum andoften with darkstripeunderneath

Variable

Humeral gland (1)raised or flat, (2)size, and (3)position

(1) Prominent andraised, (2) 2/3length of arm, and(3) centrallypositioned on theventral surface ofthe humerus

(1) Prominentand raised, (2) 2/3 length of arm,and (3) centrallypositioned on theanteroventralsurface of thehumerus

(1) Raised, (2) 1/3 to 1/2 length ofhumerus, and (3)centrallypositioned ontheanteroventralsurface of thehumerus

(1) Prominent andraised, (2) 2/3 to 3/4length of humerus (3)positioned on theanteroventral surfaceof the humerus. Maybe variable in size andposition

Dubois (1992)states suprab-rachial glandsare presentand large (notseen duringThis study)

Variable (1) Prominentand raised, (2)3/4 length ofhumerus, and(3) onanteroventralsurface

(1) Prominentand raised withdark pigment,(2) 2/3 length ofthe humerus,and (3) onanteroventralsurface

(1) Lessprominent thanSylvirana andwith darkpigment, (2) 2/3length ofhumerus, and (3)on anteroventralsurface

(1) Lessprominent thanSylvirana andwith darkpigment (2) 2/3length ofhumerus, and (3)on anteroventralsurface

Rictal ridge Weak or absent Medium to well-developed

Medium to well-developed

Very large and well-developed

Relativelylarge andbroken

Large andwell-developedand white orcream

Medium andwhite

Medium to well-developed andwhite or cream

Very large andwell-developedand white orcream

Thin and distinctor linear series ofwarts andvariable color

Upper lip coloration Gray or as rest offace

May be mottled,spotted, oruniform

Usually white Usually white; dark inA. lepus

White White andrelativelythicker thanin otherclades

White Gray, off-white,or occasionallymottled

White glandularridge on upperpart and darkmottles on lowerpart of jaw

May be gray,white,vermiculated ordark

Outer metatarsaltubercle

Absent Present and large Present or absent Present or absent Absent orsmall

Present andmedium

Present andlarge

Present andmedium

Present and large Present andmedium to large

Dorsum Shagreened andmay have a vividred or reddish-brown coloration

Mottled tospotted

Shagreened, finemottles, and mayhave small,round glandswhich may betipped withspicules

Smooth to shagreenedand uniform tomottled

Shagreened toslightly wartyand with apale or darkmid-dorsalline

Striped,mottled oruniform, andshagreened,smooth,whitespicules

Shagreened,with spiculesand uniformwith specklesor faint spots

Shagreened withspicules or maybe warty

Finely tocoarselyshagreened,sometimes withwhite spicules,and usuallymottled orspotted, but mayhave stripes

Evenlyshagreened towarty, with orwithout spicules

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Table 3 (continued)

Clade A Abavorana Clade B Pulchrana Clade CChalcorana

Clade D Amnirana Clade EHumerana

Clade FHylarana

Clade GIndosylvirana

Clade H Sylvirana Clade IHydrophylax

Clade J Papurana

Pattern on dorsalsurface of hindlimbs

Fine pale specklesor mottled

Bars with wavyedges, spotted, orvermiculated

General lack ofbars, but may befaint

Mottled or blotched,occasionally striped

Faint bars tomottled andshanks mayhave faintlines

Generally notbarred,except for thegracile ’grass’adaptedspecies.Calves mayhave faintlines

Barred on thecalf andfemur. Calvesmay haveridges or linesof spicules

Finely orcoarsely barredto mottled.Calves may havelinearly arrangedspicules

Thin, irregularbars on dorsalsurface of thighonly. Whitespicules ondorsal surface oflegs in breedingmales

Strong glandularbars to no bars.May beuniformly warty

Posterior surface ofthigh

Faintly stippled ormottled

Generallymottled, spotted,or reticulated

Same as dorsum Speckled to stronglyvermiculated

Vermiculatedto mottled

Mottled tostriped

Lightlystippled tovermiculated

Mottled orvermiculated

Stronglyvermiculated

Vermiculated tofinely mottled,but variableamong species

Body size and shape Medium androbust

Small and gracilein BA and largeand robust in BB

Small tomedium-sizedwith a long headand bullet-shaped body,limbs and bodygracile

Robust and medium tovery large

Variable insize andgracile torobust

Gracile tomedium androbust

Medium androbust

Generallymedium androbust

Robust, small tomedium-sized

Robust, mediumto extremelylarge

Flank coloration Dark brown orblack belowdorsal foldgrading to pale onventrum

Mottled orspotted, ifpattern present,or as dorsum(ground colormay be paler)

Coloration asdorsum

Variable, but usuallymottled

As dorsum ordark andmottled

Uniform tobicolor tomottled

As dorsum Dark colorationunderneathlateral ridgesfading to palewith well-defined darkspots

Strongly mottledand usually withdarkerbackground

Mostly asdorsum, but mayhave darkpatches or bemottled

Flank texture andglands

Smooth Clade BB isstrongly warty,clade BA isweakly warty

Accessoryglandular ridgesoften presentand oftenarranged linearly

Glandular or warty Smooth Smooth Shagreened orfew, scatteredwarts

Smooth or withsmall warts

Flanks may bestrongly wartyand glandular,but not arrayedin lines

May have warts

Tympanum No faint palecoloration onmargins

No faint palecoloration onmargins

No faint palecoloration onmargins

May have faint palecoloration anteriorlyand posteriorly

Faint palecolorationaroundmargins

Faint palecoloration onmargins

No faint palecoloration onmargins

Sometimes withfaint palecoloration onmargins

Large; no faintpale colorationon margins

Very small in P.jimiensis tolarge; no faintpale colorationon margins

Notes Brown or blackthroat andsometimes withsmall, pale spots(Boulenger, 1920).Was grouped withPulchrana inDubois (1992)

See Brown andGuttman (2002)for species-levelcharacters

May have many,species-specificaccessory bodyglands

Morphologically ahighly variable clade.May be highlyglandular on ventrumand have otheraccessory body glands

Pointed snout See Biju et al.(2014) forspecies-levelcharacters

Similarpostocularmasks as inPapurana

Femoral granulesare 3/4 thelength of thethigh or more

Postocular maskpresent in manyspecies

Dubois (1992) addi-tional putativecharacters

Males withoutvocal sacs (Inger,1966)

Males with orwithout pairedinternal vocalsacs

Males with orwithout pairedvocal sacs, whichdo not protrudeexternally

Males with pairedvocal sacs, which maybe internal, orprotrude externally, asreported by Channing(2001)

Malesreported tohave pairedvocal sacs,whichprotrudeexternally

Dubois statedoutermetatarsaltuberclepresent orabsent andmales lackvocal sacs

Included inSylvirana byDubois (1992)

Disc withcircum-marginalgroove issometimesabsent on finger1, and pairedvocal sacs maybe internal orexternal

Paired externalvocal sacs

Paired externalvocal sacs

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Fig. 3. Biogeographic reconstruction for Hylarana s.l. Ball-and-stick model exported from Mesquite MLSAR analysis. The black star denotes the ancestral node for the ingroupHylarana s.l. Biogeographic regions: A – Africa; B – South Asia (India, Nepal, Sri Lanka); C – Southeast Asia (Myanmar, Thailand, Cambodia, Vietnam, Laos, China, Malaysia,Java, Sumatra); D – Sulawesi; E – Philippines; F – Australasia. Numbers on nodes correspond to values in Table 4. Unnumbered nodes designate a probability of 1.00 for allthree analyses for that biogeographic region. Island groups that are not colored, but fit within the known range, are excluded because of lack of samples from that locality/region. Assigned biogeographic regions are based on range of species and collection locality when range has slight overlap across biogeographic regions.

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Although there is ambiguity in the number of colonization eventsfor the Philippines in our biogeographic reconstruction, Brownand Siler (2014) conducted a more detailed biogeographic analysisof Philippine dispersal of Hylarana s.l. and found a ‘dual-invasion’for the eastern and western arcs.

Hylarana luctuosa is the sister taxon to all other Hylarana s.l.species and has a disjunct distribution in small areas of peninsularThailand and Malaysia, Borneo, and Sumatra. As noted earlier,African and Australasian clades render Southeast Asian Hylaranas.l. paraphyletic, indicating the need for nomenclatural revision. Aparaphyletic Southeast Asian group is unsurprising inasmuch asit lies between the other biogeographic regions inhabited by theclade.

Hylarana s.l. as a taxonomic unit is uninformative as to the evo-lutionary diversification and biogeography of this large andbroadly distributed group. In the Systematic Account, we proposeto recognize ten genera, provide notes on taxonomy, and diagnoseexternal morphological characters for each genus. We expect thisapproach to stimulate further detailed morphological and taxo-nomic study on these newly defined genera.

4.2. Morphology of genera

Examination of voucher specimens and a review of the litera-ture highlighted several important morphological characters thatare potentially taxonomically informative at both the genus andspecies levels (Table 3). Here, we briefly list these characters, pri-marily to aid future studies examining widespread species orattempting to resolve relationships among species within each

Please cite this article in press as: Oliver, L.A., et al. Systematics and biogeogrtralasia, Southeast Asia, and Africa. Mol. Phylogenet. Evol. (2015), http://dx.do

genus. We stress that these observations are based solely on theliterature and our examination of a limited set of voucher speci-mens from our molecular study or the AMNH collection. Wegrouped vouchers into demonstrably monophyletic groupsretrieved in our molecular phylogeny and then attempted to makephylogenetically informed generalizations regarding the morphol-ogy of the entire clade. These observations are presented ashypotheses to be tested, and diagnoses may change with additionalwork and more extensive examination of material. This approachwas used previously for scorpions in the study of Vaejovidae(Scorpiones) and represents a useful start for approaching large,morphologically difficult taxa (González-Santillán and Prendini,2014).

Dubois (1992) considered humeral glands in males to be dis-tinct (for ‘‘Rana subsection Hydrophylax’’) in subgenera Amnirana,Humerana, Hydrophylax, Papurana, Pulchrana and Sylvirana) andindistinct or absent (for ‘‘Rana subsection Hylarana’’) for subgeneraChalcorana, Hylarana, Tylerana, and a suite of taxa of Ranidae nowrecognized at generic rank outside Hylarana s.l. (such asGlandirana, Odorrana and Sanguirana). Dubois (1992) also usedlarge humeral glands to diagnose the subgenus Humerana. Frostet al. (2006) noted that the absence of humeral glands can bedetermined only by dissection, and stated that it is not clear fromDubois (1992) whether dissections were conducted in Hylarana s.l.to ascertain this. Our examination showed that humeral glands ofsome sort were usually discernable if adult males in breeding con-dition were examined. We could not conclude that these glandswere generally absent in any subgeneric clade within Hylaranas.l. (e.g. Chalcorana, Hylarana, Tylerana). Lack of detailed homology

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Table 4Biogeographic reconstructions from MLSAR, S-DIVA, and DEC analyses. Node numberscorrespond to Fig. 3. Probabilities <0.05 are not listed. A – Africa; B – South Asia (India,Nepal, Sri Lanka); C – Southeast Asia (Myanmar, Thailand, Cambodia, Vietnam, Laos,China, Malaysia, Java, Sumatra); D – Sulawesi; E – Philippines; F – Australasia.

Node MLSAR S-DIVA DEC

Area Probability Area Probability Area Probability

1 C 0.99 CE 1.00 CE 1.002 C 0.99 C 0.50 C 0.82

CE 0.50 CE 0.183 C 0.50 CE 0.50 CE 1.00

E 0.50 E 0.504 C 0.50 CE 1.00 CE 1.00

E 0.505 C 0.99 C 0.50 C 1.00

CD 0.506 C 0.50 D 0.50 CD 1.00

D 0.50 CD 0.507 C 0.50 CD 1.00 CD 1.00

D 0.508 C 0.99 BC 1.00 BC 1.009 A 0.99 AC 1.00 AC 1.0010 C 0.99 BC 1.00 BC 1.0011 C 0.99 BC 1.00 BC 1.0012 C 0.95 CF 0.50 CF 0.82

BCF 0.50 BCF 0.1813 C 0.95 BC 1.00 BC 1.00

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assessment of these glands makes their use complicated and prob-lematic. We have here assumed that all of these variable gland con-figurations are related and homologous, because our examinationof material from all subgenera did not produce an obvious distinc-tion between them; they are all here generically called humeralglands (sensu Duellman and Trueb, 1986:59; Figs. 3–14). There isa need for a comprehensive, detailed study of body glands inHylarana s.l., and in other closely related genera of Ranidae, whichshould include dissection of material. Despite their uncertainhomology, we did note that the size and position of the humeralgland(s) along the forearm varies predictably between majorclades (Table 3). Therefore, presence or absence of a humeral gland(or brachial gland) in itself may not be a useful character to definegenera. Rather, more detailed attention needs to be paid to theexact position, shape and size of the humeral gland(s), and notewhether they fail homology tests with other similar glands, inorder to obtain any phylogenetic information of diagnostic value.Positional similarity is a well-known requirement for homologydeterminations (Patterson, 1982). Scott (2005) presented variousarguments against imprecise character-state descriptions usedverbatim from historical literature, which can adversely affectthe informativeness of morphological characters in ranoid frogs.

Another set of characters that we observed to be potentiallyinformative within these frogs is the position and shape of variousaccessory body glands in adults, particularly on the flanks andthighs, which Dubois (1992) used as diagnostic for Chalcorana.Dubois (1992) defined Hylarana s.s. as having dermal glands in lar-vae, although detailed species-level studies are few (see Gaworet al. (2009) for a regional study of Hylarana s.l. tadpoles, whereindifferences in gland position in larvae were noted among species.).Presumably, these glands persist with a fair degree of positionalsimilarity into adult frogs (there is no evidence that they fade ordisappear at metamorphosis), particularly in the generaHydrophylax (type: H. malabarica; clade I) and Hylarana s.s. (type:H. erythraea; clade F), and Amnirana (type: A. amnicola; clade D).Voucher specimens from our Amnirana and Papurana clades alsodemonstrated additional accessory body glands, which may bespecies-specific, and appear consistent within populations andputative species groups (L.A. Oliver, in prep.).

Please cite this article in press as: Oliver, L.A., et al. Systematics and biogeogrtralasia, Southeast Asia, and Africa. Mol. Phylogenet. Evol. (2015), http://dx.do

Additional external morphological characters of adults mayprove informative to species diagnoses within clades of Hylaranas.l. The position, size, and shape of the rictal gland (Inger, 1966),or clusters of glands, behind the angle of the mouth can diagnosespecies in some Hylarana s.l. complexes, sensu Inger, 1966. Wefound that whether the upper and/or lower lip was uniformlywhite, or contained patterns or blotches, was consistent withinspecies. Similarly, the general configuration of patterning presenton the lateral flanks (including presence or absence of the dark‘eye-mask’ only, or a continuous dark lateral band below the dor-solateral folds), is taxonomically useful, and has been noted previ-ously in morphological work on Papurana (Kraus and Allison,2007). Occasionally, the tympanic-border color appears as a usefultaxonomic character. The extent and degree of development of thebarring on the dorsal surface of the thigh, which can differ fromboth the barring on the dorsal surface of the shank and from thecoloration of the posterior surface of the thighs, varies withinclades and among species that we examined. The unique ‘flash’patterning on the posterior surface of thighs in Hylarana s.l. variedregularly among species and remained consistent within speciesexamined here, and may be similar to the species-specific variationin this pattern documented as being diagnostic among species ofPtychadena Boulenger, 1918 (Stewart, 1967). Particular attentionshould also be paid to the relative expansion of Digit 1 comparedto Digits 3 and 4 on the hands, which we found varied among spe-cies, while the relative size of the discs on the hands and feet var-ied among genera. Further study should be invested intodiscerning the differences in expansion of discs and presence orabsence of circummarginal grooves on certain digits in Hylarana,which was noted by Dubois (1992) and Biji et al. (2014). We hopethat future studies will investigate these character systems in moredetail within each clade.

4.3. Systematic account

Frogs currently placed in Hylarana s.l. have been apportionedamong a number of subgenera, most of these created recently(Dubois, 1992; Fei et al., 1990, 2010). Our molecular analysis iden-tifies a number of well-supported clades within Hylarana s.l. thatcorrespond in varying degrees with several of these previously pro-posed subgenera while rejecting others. Our results now allow usto better sort among available morphological evidence to supportformal recognition of several of these taxa. We believe that parti-tion of Hylarana s.l. into multiple genera using these data betterreflects the diverse biogeographic history of this large group thandoes retaining a single large genus spanning three continents.We expect it will also facilitate more-detailed investigations intothese smaller clades. In particular, we believe that the twomonophyletic invasions and radiations of Hylarana s.l. into Africaand Australasia over difficult-to-cross biogeographic barriersshould be recognized taxonomically so as to emphasize thebiological importance of those improbable events. Multipleinvasions into India from southeastern Asia are also more easilyhighlighted and discussed with this revised taxonomicframework. Recognizing the African and Papuan clades, as well asthe clades we retrieve that are clearly diagnosable morphologi-cally, requires us to also taxonomically recognize a few additionalclades that are more morphologically variable but supported byour molecular evidence so as to avoid leaving a paraphyleticHylarana. Hence, we divide Hylarana s.l. into ten genera based ona combination of our monophyletic groupings, morphological diag-nosability, biogeographical importance, and taxonomic precedence(Tables 1 and 3).

We raise Amnirana, Chalcorana, Humerana, Pulchrana,Papurana, and Sylvirana (Dubois, 1992), and Hydrophylax(Fitzinger, 1843) to generic rank. We retain Hylarana for the clade

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Fig. 4. Time-calibrated maximum clade credibility tree estimated using BEAST. Ages on nodes are in millions of years. Branch support values are BPP based on the topologyrecovered from the BEAST analysis. Node values P0.95 have a black circle and nodes that are P0.75 have a white circle. Nodes <0.75 are not labeled. Terminal namesrepresent the revised taxonomy proposed in the Systematic Account. Three nodes have symbols corresponding with colonization events: large circle (Africa), square (firstcolonization of India), and star (Australasia).

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containing H. erythraea, H. macrodactyla, H. taipehensis, and H. tyt-leri. We synonymize Boulengerana, Tenuirana, and Tylerana intoSylvirana, Hylarana, and Papurana, respectively. Additionally, wedescribe two new genera: one genus monotypic for Hylarana luctu-osa, viz. Abavorana gen. nov., and the second, Indosylvirana gen.nov., containing H. flavescens plus H. aurantiaca, H. intermedia, H.milleti, H. montana, H. temporalis, and recently described Indianspecies (Biju et al., 2014). We tentatively assign untested speciesto clades based on suggestions of close relationships from the liter-ature. However, these hypotheses need to be further tested withadditional data for reliably identified voucher specimens. Webriefly describe diagnostic morphological characters of each genusbased on our voucher specimens (Table 3) and also from descrip-tions in the literature (Biju et al., 2014; Bortamuli et al., 2010;Boulenger, 1920; Brown and Guttman, 2002; Dubois, 1992; Krausand Allison, 2007). The characters described are put forward as a

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preliminary delineation and require further exploration withineach genus.

Family Ranidae Rafinesque 1814Genus Abavorana gen. nov.

ETYMOLOGY: The name is derived from the Latin avus, meaninggrandfather, the Latin prefix ab- indicating away or from (in thesense of prior to, in this case), and the Latin rana, meaning frog.The name can be interpreted, thus, as ancestral frog and is inrecognition of this early phylogenetic separation from otherfrogs within our study clade.TYPE SPECIES: Limnodytes luctuosus Peters, 1871 by monotypy.MATERIAL EXAMINED: Abavorana luctuosa (FMNH 273219).DIAGNOSIS: Abavorana can be diagnosed from other Hylaranas.l. by having the unique combination of absence of a vocal

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sac, small pale spots on body and limbs, shagreened dorsum,red coloration on the dorsum, and absence of rictal ridges anddorsolateral folds.TAXONOMIC NOTES: Additional description of A. luctuosa inBoulenger (1920). This species was earlier assigned toPulchrana by Dubois (1992), but has been found here and inother studies to represent a separate lineage sister to remainingtaxa in Hylarana s.l. (Pyron and Wiens, 2011; Wiens et al., 2009).RANGE: Disjunct distribution in peninsular Thailand, Malaysia,Sumatra, and Borneo (Frost, 2014).

Genus Amnirana Dubois, 1992 stat. nov.:

TYPE SPECIES: Hylarana amnicola Perret, 1977 by originaldesignation.DEFINITION: All descendants of the most recent commonancestor of A. nicobariensis and A. amnicola. By implication, theAfrican taxa Hylarana asperimma, H. fonensis, H. lemairei, H. long-ipes, H. occidentalis, and H. parkeriana are provisionally includedin Amnirana.MATERIAL EXAMINED: Amnirana amnicola (AMNH 117606,117621, 122818), A. galamensis (AMNH 23543, MVZ 234148,245225), A. lepus (CAS 249985, 249987, USNM 584220), andA. nicobariensis (MVZ 239177, FMNH 266995).TAXONOMIC NOTES: We recognize this clade based on itsmonophyletic status and biogeographic cohesion and isolation.Data available at this time do not allow for a morphologicaldiagnosis due to high variability within the clade. Dubois(1992) placed the African taxa into two different sections ofhis classification of Hylarana s.l. based on the presence orabsence of distinct humeral glands. However, the body glandsin this genus are highly variable among species and, hence, can-not serve to define this clade. Another character previously con-sidered informative at higher taxonomic levels is expansion ofthe tips of the fingers into discs, and character states of thisare highly variable in Amnirana, and plesiomorphic in ranids(Scott, 2005). Furthermore, whether the posterior section ofthe abdominal skin is smooth or granular alternately occurs indifferent species of Amnirana (and differs across other cladesof Hylarana s.l.) These variable characters partially explainwhy African Hylarana s.l. was placed into two sections inDubois’ (1992) classification. Dubois (1992) placed A. galamen-sis with H. malabarica in Hydrophylax due to similar unex-panded discs. We did not find the Hydrophylax clade to beclosely related to Amnirana in our molecular analysis. Weincluded only five of 11 described species of Amnirana, andincluded Biju et al. (2014)’s Indian H. malabarica sequencesfrom GenBank. Our molecular analysis could not refute themonophyly of African Amnirana, nor the position of A. nico-bariensis (previously placed in Sylvirana) as sister to thisAfrican clade. We are hesitant to raise A. nicobariensis to sepa-rate generic status, given that the non-molecular synapomor-phies of Amnirana are not clear and external morphologyprovides no diagnostic characters at present. We concede thatthere is a huge biogeographic gap between A. nicobariensis’ dis-tribution and the rest of Amnirana. We defer any decision ofexcluding A. nicobariensis from Amnirana until further data areavailable to support a compelling decision. The range of varia-tion in tadpole characters listed in Bortamuli et al. (2010) doesnot assist in explaining the placement of A. nicobariensis withthe African species, but it is consistent with this placement.RANGE: Western and central sub-Saharan Africa, and southernportions of the Horn of Africa, associated there with CentralAfrican Forests (Frost, 2014). Amnirana nicobariensis occurs inthe Nicobar Islands, Peninsular Thailand, Sumatra, Java,Borneo, Sulu Archipelago, and Palawan (Frost, 2014).

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Genus Chalcorana Dubois, 1992 stat. nov.:

TYPE SPECIES: Hyla chalconotus Schlegel, 1837 by originaldesignation.DEFINITION: All descendants of the most recent commonancestor of C. macrops and C. chalconota. By implication,Hylarana crassiovis, H. kampeni and H. scutigera are also provi-sionally included in Chalcorana.MATERIAL EXAMINED: Chalcorana chalconota (AMNH 107901–11) C. eschatia (FMNH 268851, 268859), C. macrops (MVZ254478), C. megalonesa (FMNH 235641, 268981), C. parvaccola(FMNH 268613), and C. rufipes (FMNH 26857, 268579, 268587).DIAGNOSIS: Chalcorana can be diagnosed as the only genusof Hylarana s.l. with 1st finger 6 2nd finger, disc size on fin-gers P 2x width of the finger, and humeral gland 1/3 to 1/2length of humerus. Additionally, they have a gracile body shapeand many accessory body glands.RANGE: Southern and Peninsular Thailand, Java, Sumatra, WestMalaysia, northern and western Borneo, and Sulawesi (Frost,2014).

Genus Humerana Dubois, 1992 stat. nov.:

TYPE SPECIES: Rana humeralis Boulenger, 1887 by originaldesignation.DEFINITION: All descendants of the most recent commonancestor of H. sp. 1 (USNM 583186) and H. humeralis. By impli-cation, H. miopus and H. oatesii are also provisionally included inHumerana.MATERIAL EXAMINED: Humerana oatesii (AMNH 45579), H. sp.1 (USNM 583186), and H. sp. 2 (USNM 583170).DIAGNOSIS: Humerana can be diagnosed by its unique combina-tion of having a mid-dorsal color line, 1st finger > 2nd finger, anddisc expansion roughly equal to that of the width of the fingers.RANGE: Myanmar, Peninsular Thailand and Malaysia, north-eastern India, Nepal, Bangladesh, and Bhutan (Frost, 2014).

Genus Hydrophylax Fitzinger, 1843, stat. nov.:

TYPE SPECIES: Rana malabarica Tschudi, 1838 by originaldesignation.DEFINITION: All descendants of the most recent commonancestor of H. leptoglossa and H. malabarica.MATERIAL EXAMINED: Hydrophylax gracilis (AMNH 76991–93,77496, 74235–37, 74281–82, 76990, 77497, 83646),H. malabarica (AMNH 84587, 89797, 38080–84, 38086–89,40055–67, 63507–08), and H. leptoglossa (AMNH 53080, CAS239886).DIAGNOSIS: Hydrophylax can be diagnosed by its unique combi-nation of having a postocular mask (not as distinct as inPapurana), robust body, rear of thighs with strong vermicula-tions, large rictal gland, prominent humeral glad (but not asprominent as in morphologically similar Sylvirana), andcircum-marginal grooves sometimes absent on finger 1.RANGE: Sri Lanka, India, Bangladesh, southern Myanmar, andwestern Thailand (Frost, 2014).

Genus Hylarana Tschudi, 1838 stat. nov.:

TYPE SPECIES: Hyla erythraea Schlegel, 1837 by originaldesignation.DEFINITION: All descendants of the most recent commonancestor of H. taipehensis and H. erythraea.SYNONYMS: Tenuirana Fei et al., 1990 syn. nov. Type speciesRana taipehensis Fei et al., 1990.

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MATERIAL EXAMINED: Hylarana erythraea (AMNH 168529,168530, FMNH 263289), H. macrodactyla (AMNH 26221,180616–732, FMNH 255186, USNM 583138, 583140), H.taipehensis (AMNH 163972–73, 168753–54), and H. tytleri(CAS 247465, USNM 583188, 583190).DIAGNOSIS: Hylarana can be diagnosed by its unique combina-tion of lacking a mid-dorsal color line (present in morphologi-cally similar Humerana), 1st finger subequal to second, anddisc expansion of 1.2 to 1.7x the width of the finger.TAXONOMIC NOTES: H. macrodactyla and H. taipehensis (thetwo species previously placed in Tenuirana) were consideredto be part of Hylarana s.s. by Dubois (1992).RANGE: Bangladesh, India, Nepal, Bhutan, Cambodia, Laos,Peninsular Malaysia, Myanmar, Thailand, Vietnam, Java,Penang Perak, Borneo, Singapore, Taiwan, and southern China(Frost, 2014). Hylarana s.s. (H. erythraea) has also been intro-duced to the Philippines (Diesmos et al., 2002).

Genus Indosylvirana gen. nov.

ETYMOLOGY: The generic name is Latin, in recognition that thegeographic range of the clade is largely restricted to India andthat all included species were formerly assigned to Sylvirana.TYPE SPECIES: Rana flavescens Jerdon, 1853.DEFINITION: All descendants of the most recent commonancestor of I. milleti and I. flavescens.MATERIAL EXAMINED: Indosylvirana aurantiaca (AMNH 78924–25, 80086–67) and I. temporalis (AMNH 74217–18, 76988–89,77490–95).DIAGNOSIS: Indosylvirana can be diagnosed by its unique com-bination of having a postocular mask (faded and not as distinctas in Papurana), thin and well-defined dorsolateral folds, andprominent humeral gland extending along 3=4 length of arm.TAXONOMIC NOTES: We were unable to examine voucher spec-imens of I. milleti, and the diagnosis does not include informa-tion for this species. All species of Indosylvirana werepreviously assigned to Sylvirana by Dubois (1992) but are foundto be a separate clade in our analysis.RANGE: All species except I. milleti are restricted to Indiaand Sri Lanka. Indosylvirana milleti is located in southernVietnam, southern Thailand, and southwestern Cambodia(Frost, 2014).

Genus Papurana Dubois, 1992 stat. nov.:

TYPE SPECIES: Rana papua Lesson, 1826 by original designation.DEFINITION: All descendants of the most recent commonancestor of P. daemeli and P. papua. By implication, NewGuinean taxa H. grisea and H. novaeguineae are provisionallyincluded in Papurana as all other New Guinean species ofHylarana s.l. are representatives of Papurana, and these speciesare morphologically similar to the remaining members of thegenus. Dubois (1992) also placed H. elberti, H. florensis, and H.moluccana in Papurana. This hypothesis needs further testingusing both molecular and morphological data, but we provi-sionally include them in Papurana.SYNONYMS: Tylerana Dubois, 1992 syn. nov. Type species Ranajimiensis Tyler, 1963.MATERIAL EXAMINED: Papurana arfaki (AMNH 79933–34,191673), P. daemeli (AMNH 74863–68, 81292–93), P. garritor(AMNH 131003), P. jimiensis (AMNH 84583) P. kreffti (AMNH35404), P. novaeguineae (AMNH 84566), P. papua (AMNH98992–93), and P. supragrisea (AMNH 66616).DIAGNOSIS: Papurana can be diagnosed by the unique combina-tion of having a postocular eye mask, robust body shape, rear of

Please cite this article in press as: Oliver, L.A., et al. Systematics and biogeogrtralasia, Southeast Asia, and Africa. Mol. Phylogenet. Evol. (2015), http://dx.do

thighs with strong vermiculations, and dorsolateral folds eitherabsent or thin, with asperities.TAXONOMIC NOTES: Kraus and Allison (2007) recorded variouscharacters to distinguish among species of New GuineanHylarana. Papurana jimiensis and P. arfaki were originallyassigned to Tylerana by Dubois (1992). The greatest morpholog-ical variation in this genus exists between (P. arfaki + P. jimien-sis) and all other New Guinean species of Papurana.RANGE: New Guinea; D’Entrecasteaux Islands; Sudest,Louisiade Islands; Aru Islands; New Hanover Island; NewBritain; Yapen; Seram; Manus; Waigeo; Solomon Islands; NewIreland; Cape York Peninsula, northeastern Queensland,Australia; and northeastern border of the Gulf of Carpentaria,Northern Territory, Australia (Frost, 2014). The ranges of theprovisionally included species (P. elberti, P. florensis, and P.moluccana) include Flores, Sumba, Timor, Wetar, Babar,Tanimbar, Lombok, and Moluccas.

Genus Pulchrana Dubois, 1992 stat. nov.:

TYPE SPECIES: Polypedates signatus Günther, 1872 by originaldesignation.DEFINITION: All descendants of the most recent commonancestor of P. baramica and P. signata. By implication, H. cen-tropeninsularis and H. debussyi are also provisionally includedin Pulchrana.MATERIAL EXAMINED: Pulchrana baramica (AMNH 90514–17;FMNH 248217, 266574, 266927), P. glandulosa (AMNH 90542–49), P. picturata (FMNH 245786, 266946), P. signata (AMNH90592–99, FMNH 273117, 269721), and P. similis (FMNH266275).DIAGNOSIS: Pulchrana can be diagnosed by its unique combina-tion of weakly or strongly warty skin; a mottled to spotted dor-sum, sometimes with bright coloration; fine or wartydorsolateral folds, also sometimes with bright coloration; anda large outer metatarsal tubercle.TAXONOMIC NOTES: Brown and Guttman (2002) previouslyexamined the Pulchrana signata complex using morphologyand molecules. With the exception of Abavorana luctuosa, ourstudy supports the original delineation of Pulchrana (Dubois,1992).RANGE: Southern Vietnam, Peninsular Thailand, PeninsularMalaysia, Java, Borneo, Siberut Island, Sumatra, Singapore,Bangka Island, Natuna Islands, Sulu Archipelago, and thePhilippines (Frost, 2014).

Genus Sylvirana Dubois, 1992 stat. nov.:

TYPE SPECIES: Lymnodytes nigrovittatus Blyth, 1856 by originaldesignation.DEFINITION: All descendants of the most recent commonancestor of S. spinulosa and S. nigrovittata. Hylarana hekouensisand H. menglaensis were described as part of the S. nigrovittatagroup (Fei et al., 2008) and are also provisionally placed inSylvirana. We were unable to include in our analyses severalother mainland Southeast Asian and Indian species that havealso been placed in Sylvirana. For these species, it is necessaryto further test their taxonomic placement. It is likely, forinstance, than one or more (especially among the Indian spe-cies) may be more closely related to Indosylvirana orHydrophylax. These species are H. attigua, H. celebensis, H. chit-wanensis, H. garoensis, H. lateralis, H. latouchii, H. margariana,and H. montivaga. We consider these incertae sedis.SYNONYMS: Boulengerana Fei et al., 2010 syn. nov. Type speciesRana guentheri Boulenger, 1882.

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MATERIAL EXAMINED: Sylvirana cubitalis (CAS 210634, FMNH265818, 270736), S. guentheri (AMNH 16190, 161462–63,163940–42), S. mortenseni (FMNH 263303), S. nigrovittata(AMNH 161270–75, USNM 583124–25, 583178), and S. spinu-losa (MVZ 236683).DIAGNOSIS: Similar to Indosylvirana, Hydrophylax, andPapurana, Sylvirana can be diagnosed by its unique combinationof having a postocular eye mask, robust body shape, rear ofthighs with strong vermiculations, thick dorsolateral folds,and a humeral gland that is less prominent than that seen inIndosylvirana, but more prominent than those in Hydrophylaxand Papurana. It can be differentiated from Papurana based onthicker and better-developed dorsolateral folds, less developedpostocular mask, and more prominent humeral gland; fromHydrophylax by its smaller rictal ridge, generally larger discson the fingers, and more prominent humeral gland; and fromIndosylvirana by its thicker dorsolateral folds and less promi-nent humeral gland.TAXONOMIC NOTES: Sylvirana guentheri was the only speciesassigned to Boulengerana. We synonomize it with Sylviranabecause it falls within that clade.RANGE: Mainland China, Hainan Island, Taiwan, Myanmar,Thailand, Laos, Cambodia, Vietnam, Bhutan, Nepal,Bangladesh, and West Bengal (Frost, 2014).

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Acknowledgments

We thank the Sackler Institute for Comparative Genomics atAMNH for providing facilities and support during this research.LAO thanks her committee members George Amato (AMNH),Susan Perkins (AMNH) and Frank Burbrink (CUNY); and JohnDenton, Sara Ruane, and Rachel Welt (AMNH) for helpful

Appendix A

Accession No. Genus New Genus Species

FMNH 256531 Babina NA chapaensisFMNH 256532 Babina NA chapaensisCAS 207510 Babina NA pleuradenCIB-HUI040003 Glandirana NA minimaNIBRAM0000000445 Glandirana NA rugosaFMNH 273219 Hylarana Abavorana luctuosaUTA 44664 Hylarana Amnirana albolabris 1TMSA 84177 Hylarana Amnirana albolabris 1ROM 19861 Hylarana Amnirana albolabris 2ROM 19863 Hylarana Amnirana albolabris 2CAS 229991 Hylarana Amnirana albolabris sp.

AMNH 117606 Hylarana Amnirana amnicolaAMNH 117621 Hylarana Amnirana amnicolaAMNH 122818 Hylarana Amnirana amnicolaPEM A6989 Hylarana Amnirana darlingiMVZ 234148 Hylarana Amnirana galamensisMVZ 245225 Hylarana Amnirana galamensisCAS 249985 Hylarana Amnirana lepusCAS 249987 Hylarana Amnirana lepusUSNM 584220 Hylarana Amnirana lepusFMNH 266995 Hylarana Amnirana nicobariensisFMNH 266996 Hylarana Amnirana nicobariensisMVZ 239177 Hylarana Amnirana nicobariensisMVZ 253938 Hylarana Amnirana nicobariensis

Please cite this article in press as: Oliver, L.A., et al. Systematics and biogeogrtralasia, Southeast Asia, and Africa. Mol. Phylogenet. Evol. (2015), http://dx.do

discussion of this dissertation research. We thank David Kizirian,Margaret Arnold, David Dickey and Lauren Vonnahme for collec-tions assistance at AMNH. This manuscript was improved by com-ments from Sara Ruane and two anonymous reviewers. We wouldlike to thank all the field workers who accessioned their tissues inpublic collections (without which a large-scale study such as thiswould be impossible), as well as the curators and collections staffwho oversee the care and dissemination of these tissues andvouchers, and provided loans, including: Julie Feinstein (AmbroseMonell Cryocollection, AMNH, New York); Lauren Vonnahme,David Dickey, Margaret G. Arnold and Rob Pascocello (AMNH,New York); Jens Vindum, David Blackburn, and Robert Drewes(CAS, San Francisco); Molly Hagemann and Allen Allison (BPBM,Honolulu); Alan Resetar and Robert F. Inger (FMNH, Chicago);Donna Dittmann, Eric Rittmeyer, and Christopher Austin (LSUMZ,Baton Rouge); Carol Spencer and Jimmy McGuire (MVZ,Berkeley); Addison Wynn and Kevin de Queiroz (NMNH,Washington, DC); Bill Branch and Gillian Watson (PEM, PortElizabeth); Amy Lathrop and Bob Murphy (ROM, Ontario); andRainer Günther, Frank Tillack and Mark-Oliver Rödel (ZMB,Berlin); and Jonathan Campbell and Dwight Lawson (UTA,Arlington). This research was Supported by a National ScienceFoundation Graduate Research Fellowship to L.A. Oliver; theNational Science Foundation BIO-DEB 1021247 to E.Scott-Prendini and C.J. Raxworthy, BIO-DEB 1021299 to K.M.Kjer; BIO-DEB 103794 and BIO-DEB 743890 to F. Kraus; and theRichard Gilder Graduate School at AMNH.

Appendix A

Taxa and vouchers used in this study. Samples that wesequenced are marked as ‘‘This Study’’ and sequences we down-loaded from GenBank are cited by the original publication.

Country: Locality Study

Laos: Xieng Khouang Prov. This studyLaos: Xieng Khouang Prov. This studyChina: Yunnan Prov. This studyChina: Heilongjiang Che et al. (2007)Korea Jeong et al. (2013)Malaysia: Sarawak This studyCameroon: East Province This studyCameroon: Nguti This studyLiberia: Sapo National Park This studyLiberia: Sapo National Park This studySierra Leone: Kasewe Hills ForestReserve

This study

Cameroon: Southwest Prov. This studyCameroon: Southwest Prov. This studyCameroon: East Prov. This studyAngola: Tazua Falls This studyTanzania: Mara Region This studyGhana: Greater Accra Region This studyCameroon: Sanaga River Bank This studyCameroon: Sanaga River Bank This studyCongo: Lekoumou This studyIndonesia: West Sumatra This studyIndonesia: West Sumatra This studyIndonesia: Sumatra This studyIndonesia: Java This study

aphy of the Hylarana frog (Anura: Ranidae) radiation across tropical Aus-i.org/10.1016/j.ympev.2015.05.001

Appendix A (continued)

Accession No. Genus New Genus Species Country: Locality Study

MVZ 239431 Hylarana Chalcorana chalconota Indonesia: Sumatra This studyCAS 229564 Hylarana Chalcorana eschatia Myanmar: Pakchan Reserve Forest This studyFMNH 268851 Hylarana Chalcorana eschatia Thailand: Khao Luang National Park This studyFMNH 268859 Hylarana Chalcorana eschatia Thailand: Khao Phanom Bencha Park This studyFRIM 1539 Hylarana Chalcorana labialis Malaysia: Kedah Stuart et al. (2006)FRIM 1735 Hylarana Chalcorana labialis Malaysia: Kedah Stuart et al. (2006)MVZ 254478 Hylarana Chalcorana macrops Indonesia: Sulawesi This studyFMNH 235641 Hylarana Chalcorana megalonesa Malaysia: Sabah This studyFMNH 268981 Hylarana Chalcorana megalonesa Malaysia: Sarawak This studyMVZ 254679 Hylarana Chalcorana mocquardi Indonesia: Sulawesi This studyMVZ 254762 Hylarana Chalcorana mocquardi Indonesia: Sulawesi This studyFMNH 268613 Hylarana Chalcorana parvaccola Indonesia: West Sumatra This studyFMNH 267961 Hylarana Chalcorana raniceps Malaysia: Sarawak Stuart et al. (2006)FMNH 267962 Hylarana Chalcorana raniceps Malaysia: Sarawak Stuart et al. (2006)FMNH 268575 Hylarana Chalcorana rufipes Indonesia: West Sumatra This studySDBDU 2009.1094 Hylarana Humerrana cf. humeralis Biju et al. (2014)USNM 583186 Hylarana Humerrana sp. 1 Myanmar This studyUSNM 583170 Hylarana Humerrana sp. 2 Myanmar This studyDZ 1156 Hylarana Hydrophylax gracilis Sri Lanka: Ganemulla Biju et al. (2014)DZ 1164 Hylarana Hydrophylax gracilis Sri Lanka: Hiyare Biju et al. (2014)CAS 239886 Hylarana Hydrophylax leptoglossa Myanmar: Kyaukpyu District This studyBNHS 5879 Hylarana Hydrophylax malabarica India: Meladoor Biju et al. (2014)BNHS 5880 Hylarana Hydrophylax malabarica India: Amboli Biju et al. (2014)FMNH 263289 Hylarana Hylarana erythraea Cambodia: Koh Kong Prov. This studyFMNH 255186 Hylarana Hylarana macrodactyla Laos: Champasak Prov. This studyUSNM 583138 Hylarana Hylarana macrodactyla Myanmar This studyUSNM 583140 Hylarana Hylarana macrodactyla Myanmar This studyAMNH 168754 Hylarana Hylarana taipehensis Vietnam: Lao Cai This studyAMNH 163973 Hylarana Hylarana taipehensis Vietnam: Ha Giang This studySDBDU 2009.421 Hylarana Hylarana tytleri sp. India Biju et al. (2014)CAS 229614 Hylarana Hylarana tytleri Myanmar: Tanintharyi Div. This studyCAS 247465 Hylarana Hylarana tytleri Myanmar: Tanintharyi Div. This studyUSNM 583190 Hylarana Hylarana tytleri Myanmar This studyUSNM 583188 Hylarana Hylarana tytleri Myanmar This studyBNHS 5811 Hylarana Indosylvirana aurantiaca India: Chathankod Biju et al. (2014)SDBDU 2011.520 Hylarana Indosylvirana aurantiaca India:Chathankod Biju et al. (2014)BNHS 5842 Hylarana Indosylvirana caesari India: Humbarli Biju et al. (2014)SDBDU 2004.4527 Hylarana Indosylvirana caesari India: Amboli Biju et al. (2014)BNHS 5815 Hylarana Indosylvirana doni India: Padagiri Biju et al. (2014)BNHS 5818 Hylarana Indosylvirana doni India: Parambikulam Biju et al. (2014)BNHS 5844 Hylarana Indosylvirana flavescens India: Settukunnu Biju et al. (2014)BNHS 5845 Hylarana Indosylvirana flavescens India: Sairandhri Biju et al. (2014)BNHS 5848 Hylarana Indosylvirana indica India: Charmadi Ghats Biju et al. (2014)BNHS 5855 Hylarana Indosylvirana indica India: Meenmutty Biju et al. (2014)BNHS 5832 Hylarana Indosylvirana intermedia India: Kalpetta Biju et al. (2014)BNHS 5836 Hylarana Indosylvirana intermedia India: Kakkayam Biju et al. (2014)BNHS 5857 Hylarana Indosylvirana magna India: Pandimotta Biju et al. (2014)SDBDU 2002.2050 Hylarana Indosylvirana magna India: Kakkachi Biju et al. (2014)ROM 34429 Hylarana Indosylvirana milleti Vietnam: Gia Lai This studyBNHS 5862 Hylarana Indosylvirana montana India: Bhagamandala Biju et al. (2014)BNHS 5865 Hylarana Indosylvirana montana India: Bygoor Biju et al. (2014)DZ 1144 Hylarana Indosylvirana serendipi Sri Lanka: Kudawa Biju et al. (2014)DZ 1145 Hylarana Indosylvirana serendipi Sri Lanka: Kudawa Biju et al. (2014)BNHS 5869 Hylarana Indosylvirana sreeni India: Kuddam Biju et al. (2014)BNHS 5871 Hylarana Indosylvirana sreeni India: Kaikatti Biju et al. (2014)DZ 1141 Hylarana Indosylvirana temporalis Sri Lanka: Kudawa Biju et al. (2014)DZ 1153 Hylarana Indosylvirana temporalis Sri Lanka: Panwila Biju et al. (2014)BNHS 5837 Hylarana Indosylvirana urbis India: Kadavanthra Biju et al. (2014)BNHS 5841 Hylarana Indosylvirana urbis India: Meladoor Biju et al. (2014)BPBM 19463 Hylarana Papurana arfaki Papua New Guinea: Central Prov. This studyRG 7637 Hylarana Papurana arfaki Indonesia: Papua Prov., Wondiwoi Mts This study

(continued on next page)

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Appendix A (continued)

Accession No. Genus New Genus Species Country: Locality Study

RG 6548 Hylarana Papurana aurata Indonesia: Papua Prov., Nabire This studyRG 7588 Hylarana Papurana aurata Indonesia: Papua Prov., Nabire This studyBPBM 36024 Hylarana Papurana daemeli Papua New Guinea: East Sepik Prov. This studyBPBM 36025 Hylarana Papurana daemeli Papua New Guinea: East Sepik Prov. This studyBPBM 15488 Hylarana Papurana garritor Papua New Guinea: Gulf Prov. This studyBPBM 15489 Hylarana Papurana garritor Papua New Guinea: Gulf Prov. This studyBPBM 22831 Hylarana Papurana jimiensis Papua New Guinea: West Sepik Prov. This studyBPBM 22832 Hylarana Papurana jimiensis Papua New Guinea: West Sepik Prov. This studyBPBM 22402 Hylarana Papurana kreffti Papua New Guinea: East New Britain This studyBPBM 22403 Hylarana Papurana kreffti Papua New Guinea: East New Britain This studyBPBM 15749 Hylarana Papurana milneana Papua New Guinea: Milne Bay Prov. This studyBPBM 16361 Hylarana Papurana milneana Papua New Guinea: Milne Bay Prov. This studyBPBM 20741 Hylarana Papurana milneana Papua New Guinea: Milne Bay Prov. This studyBPBM 22844 Hylarana Papurana papua Papua New Guinea: West Sepik Prov. This studyLSUMZ 97639 Hylarana Papurana papua Papua New Guinea: Madang Prov. This studyBPBM 24218 Hylarana Papurana supragrisea Papua New Guinea: Central Prov. This studyBPBM 39587 Hylarana Papurana supragrisea Papua New Guinea: Northern Prov. This studyRG 7636 Hylarana Papurana volkerjane Indonesia: Papua Prov., Wondiwoi Mts This studyRG 7724 Hylarana Papurana volkerjane Indonesia: Papua Prov., Wondiwoi Mts This studyBPBM 16384 Hylarana Papurana waliesa Papua New Guinea: Milne Bay Prov. This studyBPBM 16398 Hylarana Papurana waliesa Papua New Guinea: Milne Bay Prov. This studyZRC8326 Hylarana Pulchrana banjarana Malaysia: Peninsular, Pahang Brown and Siler,

2014FMNH 248217 Hylarana Pulchrana baramica Brunei: Belait District This studyFMNH 266574 Hylarana Pulchrana baramica Malaysia: Sarawak This studyFMNH 266927 Hylarana Pulchrana baramica Indonesia: West Sumatra This studyFMNH 248254 Hylarana Pulchrana glandulosa Brunei: Belait District Brown and Siler

(2014)FMNH 266573 Hylarana Pulchrana glandulosa Malaysia: Sarawak Stuart (2008)KU 302375 Hylarana Pulchrana grandocula Philippines: Camiguin Sur Island Brown and Siler

(2014)KU 302378 Hylarana Pulchrana grandocula Philippines: Camiguin Sur Island Brown and Siler

(2014)KUHE 17593 Hylarana Pulchrana laterimaculata Malaysia: Sarawak Matsui et al. (2012)KU 303577 Hylarana Pulchrana mangyanum Philippines: Mindoro Island Brown and Siler

(2014)KU 303578 Hylarana Pulchrana mangyanum Philippines: Mindoro Island Brown and Siler

(2014)ELR 164 Hylarana Pulchrana melanomenta Philippines: Tawi-tawi Island Brown and Siler

(2014)ELR 165 Hylarana Pulchrana melanomenta Philippines: Tawi-tawi Island Brown and Siler

(2014)KU 309009 Hylarana Pulchrana moellendorffi Philippines: Palawan Island Brown and Siler

(2014)KU 327050 Hylarana Pulchrana moellendorffi Philippines: Palawan Island Brown and Siler

(2014)FMNH 245786 Hylarana Pulchrana picturata Malaysia: Sabah This studyFMNH 266946 Hylarana Pulchrana picturata 2 Indonesia: West Sumatra This studyMZB Amp 14565 Hylarana Pulchrana rawa Indonesia: Sumatra Matsui et al. (2012)BJE 202 Hylarana Pulchrana siberu Indonesia: Siberut Island Brown and Siler

(2014)BJE 236 Hylarana Pulchrana siberu Indonesia: Siberut Island Brown and Siler

(2014)FMNH 273117 Hylarana Pulchrana signata Malaysia: Sarawak This studyFMNH 266275 Hylarana Pulchrana similis Philippines: Luzon, Zambales This studyCAS 210634 Hylarana Sylvirana cubitalis Myanmar: Shan State This studyFMNH 265818 Hylarana Sylvirana cubitalis Thailand: Loei, Phu Rua This studyFMNH 270736 Hylarana Sylvirana cubitalis Thailand: Nan Prov., Pua District This studyFMNH 267767 Hylarana Sylvirana faber Cambodia: Pursat Prov. Stuart (2008)AMNH 163940 Hylarana Sylvirana guentheri Vietnam: Ha Giang This studyAMNH 163941 Hylarana Sylvirana guentheri Vietnam: Ha Giang This study

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958

959960961962963964965966967968969970971972973974975976977978979980981

9829839849859869879889899909919929939949959969979989991000100110021003100410051006

Appendix A (continued)

Accession No. Genus New Genus Species Country: Locality Study

FMNH 255637 Hylarana Sylvirana maosonensis Vietnam: Nghe An This studyFMNH 263303 Hylarana Sylvirana mortenseni Cambodia: Koh Kong Prov. This studyFMNH 266318 Hylarana Sylvirana mortenseni Cambodia: Koh Kong Prov. This studyUSNM 583178 Hylarana Sylvirana nigrovittata Myanmar This studyUSNM 583124 Hylarana Sylvirana nigrovittata sp.

1Myanmar This study

USNM 583125 Hylarana Sylvirana nigrovittata sp.1

Myanmar This study

AMNH 161290 Hylarana Sylvirana nigrovittata sp.2

Vietnam: Ha Tinh This study

AMNH 161299 Hylarana Sylvirana nigrovittata sp.2

Vietnam: Ha Tinh This study

AMNH 161280 Hylarana Sylvirana nigrovittata sp.3

Vietnam: Ha Tinh This study

MVZ 236683 Hylarana Sylvirana spinulosa China: Hainan Prov. This studyROM 44390 Hylarana Sylvirana spinulosa China: Hainan Prov. This studyFMNH 268503 Limnonectes NA kochangae This studyFMNH 258107 Odorrana NA absita Laos: Xe Kong Prov. Stuart (2008)CAS 233900 Odorrana NA andersonii China: Yunnan Prov. This studyCAS 233901 Odorrana NA andersonii China: Yunnan Prov. This studyFMNH 255611 Odorrana NA bacboensis Vietnam: Nghe An Prov. Stuart (2008)CAS 207505 Odorrana NA grahami China: Yunnan Prov. Stuart (2008)SCUM0405180CJ Odorrana NA hejiangensis China: Hejiang, Sichuan Che et al. (2007)ROM 26370 Odorrana NA hmongorum Vietnam: Lao Cai Prov. Stuart (2008)FMNH 273209 Odorrana NA hosii Malaysia: Sarawak This studyFMNH 263415 Odorrana NA livida Thailand: Prachuap Kirikhan Prov. Stuart et al. (2006)FMNH 233029 Odorrana NA margaretae China: Sichuan Prov. Stuart (2008)ROM 39907 Odorrana NA morafkai Vietnam: Tram Lap Stuart (2008)No voucher Odorrana NA tormota China: Anhui Prov. Stuart (2008)CAS 245414 Rana NA japonica Myanmar: Myitkyina Dist. This studyFMNH 259496 Sanguirana NA igorata Philippines: Luzon, Kalinga Stuart (2008)USNM 512317 Sanguirana NA luzonensis Philippines: Polillo Island This studyRMB 3011 Sanguirana NA sanguinea Philippines: Palawan Bossuyt et al. (2006)

Institutional abbreviations: American Museum of Natural History (AMNH); Bombay Natural History Society Museum (BNHS); Bernice Pauahi Bishop Museum (BPBM); Ben JEvans field number (BJE); California Academy of Sciences (CAS);Chengdu Institute of Biology, the Chinese Academy of Sciences (CIBHU); Department of Zoology, University ofPeradeniya (DZ); Edmund B. Leo Rico field number (ELR); Field Museum of Natural History (FMNH); Forest Research Institute of Malaysia (FRIM); Kansas UniversityBiodiversity Institute (KU) ;Kyoto University (KUHE); Louisiana State University Museum of Natural Science (LSUMZ); Museum of Vertebrate Zoology at Berkeley (MVZ);Museum Zoologicum Bogoriense (MZB); National Institute of Biological Resources South Korea (NIBR); Rainer Günther field number (RG); Rafe M. Brown (RMB); RoyalOntario Museum (ROM); Systematics Lab, University of Delhi (SBDU); Sichuan University Museum (SCUM); Ditsong NationalMuseum of Natural History (TMSA); NationalMuseum of Natural History (USNM); University Texas at Austin (UTA); Zoological Reference Collection of the Raffles Museum, National University of Singapore (ZRC).

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